Proc. NatL Acad. Sci. USA Vol. 78, No. 10, pp. 6261-6265, October 1981 Cell Biology Fibronectin-like protein in Porifera: Its role in cell aggregation (phylogenesis/celi membrane/intercellular matrix/differentiation/morphogenesis) JACQUELINE LABAT-ROBERT*, LADISLAS ROBERT*, CAROLE AUGERt, CLAIRE LETHIASt, AND ROBERT GARRONEt *Laboratoire Biochimie du Tissu Conjonctif (GR Centre National de la Recherche Scientifique, No. 40), Facult6 de MWdecine, UniversitM Paris-Val de Marne, 8 rue du Ceneral Sarrail, 94010 Crkteil Cedex, France; and tLaboratoire Histologie et Biologie Tissulaire (LA Centre National de la Recherche Scientifique, No. 244), UniversitM Claude Bernard, 43 Bd. du 11 Novembre, 69621 Villeurbanne, France Communicated by D. H. R. Barton, June 15, 1981 ABSTRACT Experiments were carried out on a freshwater sponge cells-were used to show whether fibronectin plays a sponge (Ephydatia muderi) in order to demonstrate the presence role in this process. of fibronectin in Porifera. By using antibodies to highly purified Previous experiments carried out in our laboratory had dem- human plasma fibronectin, the presence of a similar or identical onstrated the presence of structural glycoproteins in various protein could be demonstrated in the membranes of E. mullen marine sponges (12-14). These glycoproteins were obtained in cells such as epithelial cells, fibroblast-like cells, and choanocytes. a highly purified form and were shown to have an amino acid The reaction was specific, could be abolished by the addition of composition quite similar to that found in structural glycopro- excess fibronectin, and was not observed with nonimmune rabbit from vertebrate tissues These serum. The immune fluorescent reaction became stronger when teins isolated (8-10, 15). sponge the sponge cells were pretreated with acetone and could also be glycoproteins, however, were of a relatively low molecular observed, although with a less intense staining, on the intercellular weight. matrix. This shows the predominant presence of a sponge fibro- nectin-like protein in the cell membranes and also its presence to MATERIALS AND METHODS a lesser extent in the intercellular matrix. When dissociated Materials. Human plasma CIg was purified by the method sponge cells were led to reassociate under the microscope, reas- of Vuento and Vaheri (16). The purity of the preparation was sociation could be completely inhibited by anti-human fibronectin checked by acrylamide gel electrophoresis and by antiserum up to a dilution of 1:120 and partially inhibited up to immunodiffusion. a dilution of 1:240. The reassociation of dissociated sponge cells The antibody to highly purified human plasma CIg was pre- could also be inhibited by the addition of purified gelatin but not pared in rabbits. Other immune serawere obtained through the with serum albumin or with a normal, nonimmune rabbit serum. courtesy ofA. Vaheri (Helsinki, Finland) and ofMosesson (New These results clearly indicate that a sponge cell fibronectin-like York). All three sera gave a single line with total human plasma protein may play an important role as the (or one of the) recog- which showed an identity with the line given by highly purified nition site(s) of the aggregation factor(s) and can therefore be di- plasma fibronectin. rectly involved in cell association, morphogenesis, and Gelatin from pig skin collagen type I and bovine serum al- differentiation. bumin were obtained from Sigma. Immunofluorescence. These experiments were carried out Fibronectin, cold-insoluble globulin (CIg), and large external according to the indirect Coon's technique using sheep antisera transformation sensitive protein (LETS protein) are more or less to rabbit IgG labeled with fluorescein (Pasteur Institute, Paris). synonymous expressions for a family of closely related glyco- Sponge Culture. Freshwater sponges (Ephydatia mulleri) proteins (1-4). These glycoproteins were found in blood plasma were grown from gemmules collected in local brooks near Lyon. and on the cell membranes of several different kind of mes- The sponges were cultivated in the laboratory at 20°C in a 1:1 enchymal cells such as fibroblasts as well as in the intercellular (vol/vol) mixture of soft mineral water ("Evian") and distilled matrix (5-7). water, between two glass cover slips, according to the "sand- On the basis of their amino acid and carbohydrate compo- wich" method of Ankel and Eigenbrodt (17). Some gemmules sition, their solubility behavior, and their presence in the in- were allowed to develop simply on cover slips in the same tercellular matrix, they appear to belong to the class ofstructural medium. glycoproteins (8-10). They play a role in the interaction be- Preparation of Cells for Fluorescence Microscopy. The tween cell membrane and intercellular matrix as far as they as- sponges were cultivated for 8 days. Just before use, the two sociate more or less specifically with collagen fibers. cover slips were separated so that each carried a thin layer of Collagen, in a microscopically and chemically well-recogniz- sponge tissue. Cells were exposed in sequence to dilute (100 able form, appears first during phylogenesis in Porifera or mosM) phosphate-buffered saline (PjNaCl), 3.5 % paraformal- sponges (11). It was therefore important to investigate whether dehyde in PjNaCl (for 30 min), and three 10-min rinses in P/ fibronectin is also present at this early stage of phylogenesis. NaCl. For surface staining, cells were not made permeable; for In order to demonstrate the presence of fibronectin-like pro- internal staining, cells were made permeable with acetone (18): teins in Porifera, we used antibodies prepared against human 2 min in 50% acetone at -20°C, 5 min in pure acetone at 40C, plasma CIg (1) as well as highly purified fibronectin obtained and 2 min in 50% acetone at room temperature. Antifibronectin from human plasma. Two different approaches-immune flu- antiserum was applied at 1:60 and 1:120 dilutions for 45 min. orescence and the inhibition of reassociation of dissociated Staining was indirect, with fluorescein isothiocyanate-conju- gated sheep anti-rabbit IgG. Controls were performed by re- The publication costs ofthis article were defrayed in part bypage charge payment. This article must therefore be hereby marked "advertise- Abbreviations: CIg, cold-insoluble globulin, P/NaCl, phosphate-buf- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. fered saline. 6261 Downloaded by guest on September 29, 2021 6262 Cell Biology: Labat-Robert et aPProc. Nad Acad. Sci. USA 78 (1981) placing the antiserum with one of the following: normal rabbit EDTA/34 mM NaCV1.34 mM KCV1.38 mM glucose/1.07 serum, a mixture offibronectin and antifibronectin antiserum, mM NaCOJ0.7 mM K2HPO4, buffered to pH 8 with 6 mM and PINaCl. Each treatment was followed by three 10-min Tris. rinses in PJNaCl. Cell suspensions were prepared by replacing the culture Fluorescence Microscopy. Slides were examined with a Leitz medium by the dispersion medium during 10 min. Dispersion photomicroscope equipped with epifluorescence illumination was ensured mechanically by pipetting. The suspension was (Ploemopak system). Fluorescence was excited with the output then centrifuged at 200 x g for 5 min. The supernatant was ofa high-pressure Hg lamp (50 W) filtered through a Leitz FITC discarded and the pellet was resuspended in the culture me- interference filter. Cells were observed through X25 and X40 dium. This procedure was repeated twice and the final pellet lens. was resuspended in 0.5 ml ofculture medium. A high-density Reaggregation Experiments. The dispersion medium, pre- population ofabout 2 x 107 cells per ml was obtained. In order pared according to Curtis and Van de Vyver (19), was 0.25 mM to study the interference of antifibronectin antiserum, of gel- FIG. 1. Cell cultures grown from gemmules examined with epifluorescence illumination after treatment with rabbit antifibronectin antiserum and fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG. (A) The fluorescence is located largely at the periphery (arrows) of the large ep- ithelial cells. Aperinuclear area (presumably the Golgiregion) is also stained (G). This patternoffluorescence isobviouslydifferentfromthestaining of the small motile cells which are heavily labeled (Inset). (x360; Inset, x200.) (B) Fluorescent staining of the cells ensuringthe waterflow through the sponge. (x500.) (C) Dense staining of a fibroblast-like cell (lophocyte). (X800.) Downloaded by guest on September 29, 2021 Cell Biology: Labat-Robert et al. Proc. Natd Acad. Sci. USA 78 (1981) 6263 _|| ,\-,,fflti,:~~~~~~~~*' at 4~~~~~VI _ _ Lma" 'Ei FRifioltiaeb t ) i l 3 inndt..)- DQ c nmut1i (E)hadbeade.Ian,mscelaermndsnl.(akfedeaia All. FIG. 2. Reassociation of dissociated sponge cells and its inhibition by anti-Clgantiserum and by gelatin. (A) Dissociated cells after 30 min culture in normal medium. Numerous cells have adhered into small, clumps. (Dark-field examination; x200.) (B-E) Dissociated cells after 30 min culture in medium to which 1:40 antifibronectin antiserum (B), 1% gelatin (C), 1:40 normal nonimmune rabbit serum (D), or 1% bovine serum albumin (E) had been added. In B and C, most cells have remained single. (Dark-field examination; x200.) Downloaded by guest on September 29, 2021 6264 Cell Biology: Labat-Robert et aL Proc. Nad Acad. Sci. USA 78 (1981) atin, or of bovine serum albumin with the reassociation of dis- hibited reaggregation in a manner similar to the inhibition pro- sociated sponge